Topical antibiotic formulations

ABSTRACT

There are disclosed topical silver(II) antibiotic formulations. Other embodiments are also disclosed.

This application is a continuation of U.S. Ser. No. 15/905,797, filedFeb. 26, 2018 as a continuation-in-part of U.S. Ser. No. 15/064,623,filed Mar. 9, 2016 as a continuation of PCT/IB2015/050630 filed Jan. 27,2015, which claims the benefit of U.S. Ser. No. 61/931,944, filed Jan.27, 2014. Said applications are incorporated herein by reference for allpurposes as if fully set forth herein.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to antibiotic formulations and methods,and more particularly, to topical silver(II) antibiotic formulations,methods of production thereof, and methods of use thereof.

While substantial advances have been made in the treatment of topicalwounds, both chronic and acute, the inventors believe there is a needfor further improvements in formulating stable, efficacious topicalantibiotic formulations and medical devices; the subject matter of thepresent disclosure and claims is aimed at fulfilling this need.

SUMMARY OF THE INVENTION

According to teachings of the present invention there is provided anantimicrobial formulation including: (a) at least one silver-containingcompound, including an anti-microbial agent containing an aliphaticsilver carboxylate, the silver of the aliphatic silver carboxylatehaving a nominal valence of 2, the at least one silver-containingcompound having an average valence of at least 1.1; and (b) a carrierbase; the at least one silver-containing compound being dispersed withinthe base.

According to an aspect of the present invention there is provided anantimicrobial formulation suitable for application to skin tissue, theformulation including: (a) at least one silver-containing compoundincluding at least one silver carboxylate, the silver of the carboxylatehaving a nominal valence of 2 or at least 2, the at least onesilver-containing compound having an average valence of at least 1.1;and (b) a carrier base; the at least one silver-containing compoundbeing intimately dispersed within the base; the formulation having astandard whiteness value of at least 4.0, at least 4.1, at least 4.2, orat least 4.3 reflective units (RU); the formulation having a totalsilver concentration of at least 0.10%, at least 0.20%, at least 0.30%,at least 0.50%, at least 0.70%, at least 1.00%, at least 1.5%, at least2.0%, at least 2.5%, or at least 3.0%.

According to an aspect of the present invention there is provided anantimicrobial formulation suitable for use in topical applications toskin tissue, the formulation including at least one silver-containingcompound, including an anti-microbial agent containing at least onesilver carboxylate, the silver of the carboxylate having a nominalvalence of at least 2, the at least one silver-containing compound.having an average valence of at least 1.1.

According to an aspect of the present invention there is provided a useof a silver carboxylate in the manufacture of a medicament for thetreatment of a topical condition, the silver of the silver carboxylatehaving a nominal valence of at least 1.1, at least 1.2, at least 1.3, atleast 1.4, at least 1.5, at least 1.6, at least 1.7. at least 1.8, atleast 1.9, or 2.0 or substantially 2.0.

According to an aspect of the present invention there is provided anantimicrobial formulation suitable for application to skin tissue, theformulation including: (a) at least one silver-containing compound,including an anti-microbial agent containing at least one silvercarboxylate, the silver of the silver carboxylate having a nominalvalence of at least 1.1; and (b) a carrier base.

According to an aspect of the present invention there is provided amethod of producing a silver(II) carboxylate or silver(II) carboxylateformulation, the method including the steps of: (a) mixing thecarboxylic acid and the silver(II) oxide to produce a reaction mixture;and (b) heating the reaction mixture to produce an aliphatic silvercarboxylate, the silver of the aliphatic silver carboxylate having anominal valence of 2.

The reaction mixture obtained, or the silver(II) carboxyl ate therein,may be mixed with a carrier base to produce the silver(II) carboxyl ateformulation.

The reaction temperature may be at least 50° C., at least 60° C., or atleast 70° C., and more typically, at least 80° C., at least 84° C., orat least 86° C. The reaction temperature may be at most 135° C., at most125° C., at most 115° C., at most 110° C., at most 107° C., at most 105°C., at most 103° C., at most 100° C., at most 98° C., at most 96° C., orat most 94° C. The reaction temperature may be within a range of 60° C.to 110° C., 80° C. to 110° C., 80° C. to 105° C., 84° C. to 1.05° C.,84° C. to 100° C. 84° C. to 98° C., 84° C. to 96° C., or 86° C. to 95°C.

The reaction mixture may be agitated for at least a portion of step (b),and more typically, substantially throughout step (b).

The carboxylic acid may be pre-heated, as necessary, prior to the mixingwith the silver(II) oxide. Typically, the carboxylic acid is brought toliquid form prior to this mixing.

A solvent or solvents may be introduced to form a portion of thereaction mixture. Preferably, any such solvents should be impervious orlargely impervious to oxidation by the silver(II) oxide, and may beselected to at least partially dissolve the carboxylic acid.

The heating may be curtailed after a lightening of the reaction mixture.The lightening may be visually or instrumentally observed.

According to further features in the described preferred embodiments,the formulation is in the form of a cream, an emulsion, or an ointment.

According to still further features in the described preferredembodiments, the total silver content of the formulation, or totalsilver content of the at least one silver-containing compound, is withina range of 0.0005% to 20%, 0.0005% to 12%, 0.0005% to 7%, 0.0005% to3.5%, 0.0005% to 3%, 0.0005% to 2.5%, 0.001% to 3.5%, 0.005% to 3.5%,0.01% to 3.5%, 0.03% to 3.5%, 0.05% to 3.5%, 0.10% to 3.5%, 0.30% to3.5%, 0.5% to 3.5%, 0.7% to 3.5%, or 0.9% to 3.5%, by weight.

According to still further features in the described preferredembodiments, the aliphatic carboxylate content is at least 0.1%, on anAg₄O₄ weight basis, the formulation being white or at least off-white.

According to still further features in the described preferredembodiments, the aliphatic carboxylate content is at least 0.1%, on anAg₄O₄ weight basis, the formulation is white or at least off-white.

According to still further features in the described preferredembodiments, the formulation typically has a standard whiteness value ofat least 3.4, at least 3.5, at least 3.6, at least 3.7, at least 3.8, orat least 3.9 reflective units (RU), at least within a range of 0.1% to1.7%, on an Ag₄O₄ weight basis.

According to still further features in the described preferredembodiments, the total silver content of the at bast onesilver-containing compound is within a range of 0.09% to 1.7%, on anAg₄O₄ weight basis, and the formulation has a standard whiteness valueof at least 4.0, at bast 4.1, at least 4.2, or at least 4.3 reflectiveunits (RU).

According to still further features in the described preferredembodiments, the total silver content of the at least onesilver-containing compound is within a range of 0.8% to 3.4%, by weight,and the formulation has a standard whiteness value of at least 3.5, atleast 3.6, at least 3.7, at least 3.8, or at least 3.9 reflective units(RU).

According to still further features in the described preferredembodiments, the silver carboxylate and the carrier base are selectedsuch that after standard ultraviolet light (UV) treatment, in which theformulation is subjected to constant exposure to UV for 12 hours at 240nm, the standard whiteness value of the formulation remains within 0.6RU, within 0.5 RU, within 0.4 RU, within 0.3 RU, or within 0.2 RU, ofthe initial whiteness value of the formulation prior to the treatment.

According to still further features in the described preferredembodiments, the silver carboxylate and the carrier base are selectedsuch that standard ultraviolet light (UV) treatment, in which theformulation is subjected to constant exposure to UV for 12 hours at 240nm, a post-UV whiteness value of the formulation remains at least 3.5reflective units (RU), at least 3.6 RU, at least 3.7 RU, at least 3.8RU, at least 3.9 RU, at least 4.0 RU, or at least 4.1 RU.

According to still further features in the described preferredembodiments, the formulation contains the silver carboxylate in a rangeof 0.30% to 3.5%, 0.4% to 3.5%, 0.5% to 3.5%, 0.6% to 3.5%, 0.7% to3.5%, 0.30% to 3%, 0.4% to 3%, 0.5% to 3%, or 0.6% to 3%, by weight.

According to still further features in the described preferredembodiments, the average valence is at least 1.2, at least 1.3, at least1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least1.9, or about 2.0.

According to still further features in the described preferredembodiments, the formulation contains less than 5%, less than 4%, lessthan 3%, less than 2.5%, less than 2.0%, less than 1.5%, less than 1.2%,less than 1%, less than 0.8%, less than 0.6%, or less than 0.4% of zincoxide.

According to still further features in the described preferredembodiments, the formulation contains less than 5%, less than 4%, lessthan 3%, less than 2.5%, less than 2.0%, less than 1.5%, less than 1.2%,less than 1%, less than 0.8%, less than 0.6%, or less than 0.4% of awhitening agent such as an inorganic whitening agent.

According to still further features in the described preferredembodiments, the weight ratio of whitening agent to the at least onesilver-containing compound is less than 7:1, less than 5:1, less than3:1, less than 2:1, less than 1.5:1, less than 1.2:1, less than 1.1,less than 0.8:1, less than 0.6:1, less than 0.4:1, less than 0.3:1, lessthan 0.2:1, less than 0.1:1, or less than 0.05:1.

According to still further features in the described preferredembodiments, the whitening agent includes, primarily includes, orconsists essentially of a divalent salt or oxide, such a calcium salt oroxide, or a magnesium salt or oxide.

According to still further features in the described preferredembodiments, the formulation contains less than 10%, less than 9%, lessthan 8%, less than 6%, less than 4%, less than 2%, less than 1%, lessthan 0.5%, less than 0.2%, less than 0.1%, or less than 0.05%, in total,of silver-containing compounds selected from the group consisting ofsilver(II) fluoride (AgF₂), silver(II) picolinate (C₁₂H₈AgN₂O₄) or anyother silver(II) containing compound having a nitrogen atom in the ringor aromatic ring, silver(I) oxide and silver(II) oxide.

According to still further features in the described preferredembodiments, the at least one silver carboxylate includes a divalent orat least divalent silver carboxylate having a carbon number or averagecarbon number less than 40, less than 38, less than 36, less than 34,less than 33, less than 32, less than 31, less than 30, less than 29,less than 28, less than 27, less than 25, less than 23, less than 22, orless than 21.

According to still further features in the described preferredembodiments, the at least one silver carboxylate includes a divalent orat least divalent silver carboxylate having a carbon number or averagecarbon number greater than 9, greater than 10, or greater than 11.

According to still further features in the described preferredembodiments, the carbon number or average carbon number is less than 40and greater than 11, less than 40 and greater than 13, less than 40 andgreater than 14, less than 38 and greater than 11, less than 36 andgreater than 11, less than 34 and greater than 11, less than 32 andgreater than 11, less than 31 and greater than 11, less than 30 andgreater than 11, less than 29 and greater than 11, less than 28 andgreater than 11, or less than 27 and greater than 11.

According to still further features in the described preferredembodiments, the formulation contains at least one carboxylic acid,optionally including a corresponding carboxylic acid of the silvercarboxylate having the nominal valence of at least 2, a molar ratio ofthe corresponding carboxylic acid to the silver carboxylate optionallybeing at least 0.01, at least 0.025, at least 0.05, at least 0.1, atleast 0.2, at least 0.5, at least 1.0, at least 1.5, at least 2, or atleast 3.

According to still further features in the described preferredembodiments, the hydrocarbon structure of the carboxylate is selectedfrom at least one of the group consisting of fully saturated,monounsaturated, and polyunsaturated structures.

According to still further features, the backbone structure of thecarboxylate includes at least one structure selected from the groupconsisting of a straight hydrocarbon chain and a branched hydrocarbonchain.

According to still further features, the backbone structure of thecarboxylate includes at least one structure selected from the groupconsisting of a ring structure and an aromatic structure.

According to still further features, the carboxylate includes a silvercarboxylate of a keto-carboxylic acid.

According to still further features, the formulation has, at a viscosityof at least 25 cP, at least 100 cP, at least 250 cP, at least 500 cP, atleast 1,000 cP, at least 5,000 cP, at least 20,000 cP, at least 50,000cP, at least 150,000 cP, at least 500,000 cP, at least 1,000,000 cP, atleast 3,000,000 cP, or at least 10,000,000 cP.

According to still further features, the formulation has a particulartotal silver content, and the concentration of elemental silver withinthe formulation is at most 50%, at most 30%, at most 15%, at most 5%, atmost 3%, or at most 1% of the particular total silver content.

According to still further features. the carrier base including orpredominantly including an oleaginous material that may include orpredominantly include a material selected from the group consisting ofbeeswax, petrolatum, a liquid wax ester, an oil, and a polyethylene wax.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice. Throughout thedrawings, like-referenced characters are used to designate likeelements.

In the drawings:

FIG. 1 provides an Electron Spin Resonance (ESR) spectrum for thereaction product of Example 4;

FIG. 2 provides a comparative ESR spectrum of Ag(II) picolinate;

FIG. 3 provides an infra red (IR) spectroscopy plot of the reactionproduct of Example 6, vs. the spectrum of the palmitic acid feedmaterial;

FIG. 4 is a bar graph showing the anti-bacterial performance, over time,of aliphatic silver(II) carboxylates of Examples 18-29, usingStaphylococcus aureus 25923;

FIG. 5 is a bar graph showing the antibacterial performance, over time,of aliphatic silver(II) carboxylates of Examples 18-29, usingEscherichia coli 35218;

FIG. 6 is a bar graph showing the anti-fungal performance, over time, ofaliphatic silver(II) carboxylates of Examples 18-29, using Candidaalbicans 10231;

FIG. 7 provides an ESR spectrum for a mixture of silver distearate (90%)and silver dibehenate (10%);

FIG. 8 is a bar graph of formulation whiteness for formulationscontaining: (1): Ag₄O₄ (0.7%); 2): Ag₂O (0.7%); and (4): Ag(II)picolinate (0.7%), each in a base containing beeswax (19.8%) and jojobaoil (79.5%), vs. an inventive formulation containing 0.7% silverdistearate in a substantially identical base;

FIG. 9 is a bar graph of formulation whiteness for prior-art silver(II)oxide formulations containing: (1): Ag₄O₄ (0.1%); (2): Ag₂O (0.7%); (3):Ag₂O (1.5%); and (4): Ag₄O₄ (3%), before and after being subjected to UVexposure o;

FIG. 10 is a bar graph of formulation whiteness for prior-art silver(I)oxide formulations containing: (1): Ag₂O (0.1%); (2): Ag₂O (0.7%); (3):Ag₂O (1.5%); and (4): Ag₂O (3%), before and after being subjected to UVexposure;

FIG. 11 is a bar graph of formulation whiteness for inventiveformulations produced from Ag₄O₄ and beeswax, before and after beingsubjected to UV exposure;

FIG. 12 is a bar graph of formulation whiteness for prior-artformulations containing Ag₄O₄ and various inorganic whiteners, beforeand after being subjected to UV exposure;

FIG. 13 is a bar graph of formulation whiteness for inventiveformulations produced from Ag₄O₄ and beeswax, before and after beingsubjected to UV exposure, some of the formulations containing variousinorganic whiteners; and

FIG. 14 is a bar graph of formulation whiteness for inventiveformulations containing a silver(II) carboxylate in various bases,before and after being subjected to UV exposure.

DETAILED DESCRIPTION

The principles of the silver(II) carboxylate formulations, methods ofproducing these formulations, and methods of use thereof, in accordancewith the present invention, may be better understood with reference tothe figures and the accompanying description.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description. The invention maybe capable of other embodiments or of being practiced or carried out invarious ways. Also, it is to be understood that the phraseology andterminology employed herein is for the purpose of description and shouldnot be regarded as limiting.

Ag₄O₄ (tetrasilver tetroxide) may be particularly reactive with respectto other components in topical formulations, thereby compromising thestability of the formulation. The formulation may then acquire,disadvantageously, a dark brown or black color.

The inventors have discovered a method of preparing silver(II)carboxylate formulations having anti-microbial properties. Many of suchformulations may advantageously have a light (e.g., light yellow, tan,or off-white) or white appearance.

Strikingly, and in sharp contrast to formulations containing silver(II)oxide and silver(I) oxide, the formulations of the present invention mayexhibit no darkening, or at most a slight darkening with increasingconcentration of the silver-containing anti-microbial agent.

The silver(II) carboxylates in the inventive formulations may typicallyhave a nominal valence of 2, or at least 2. The concentration of thesesilver(II) carboxylates within the formulations may be at least 20 ppm,and up to 15% or up to 20% by weight, or inure. In some embodiments theconcentration of these silver(II) carboxylates is at least 30 ppm, atleast 100 ppm, at least 0.05%, at least 0.10%, at least 0.3%, at least0.5%, at least 0.7%, at least 1%, at least 2%, at least 3%, at least 5%,at least 7%, at least 10%, at least 15%, or at least 20%. It must beemphasized that concentrations as low as 20 ppm have been found to behighly efficacious, from an anti-microbial or antibiotic standpoint.

In some embodiments, the concentration of these silver carboxylates isat most 10%, at most 7%, at most 5%, at most 3.5%, at most 3%, or atmost 2.5%. In some embodiments, the concentration of these silvercarboxylates is within a range of 20 ppm to 50%, 20 ppm to 45%, 20 ppmto 40%, 20 ppm to 30%, 20 ppm to 20%, 20 ppm to 15%, 20 ppm to 10%, 20ppm to 5%, 100 ppm to 3%, 0.05% to 3%, 0.1% to 3%, 0.2% to 3%, 0.3% to3%, 0.5% to 3%, or 0.7 to 3%.

Formulations of the present invention have been fund to possessantibiotic properties, i.e., they may attack and destroy at least onespecies or type of microorganism, while selectively exhibiting relativeinertness with respect to human and mammalian cells. More typically, theantibiotic substance selectively attacks and destroys at least onespecies or type of microorganism that commonly populates the skin,surface wounds, bedsores and the like, while exhibiting relativeinertness, with respect to skin cells of humans and mammals.

EXAMPLES

Reference is now made to the following examples, which together with theabove description, illustrate the invention in a non-limiting fashion.

In these examples, the microbes used: Escherichia coli 35218 andStaphylococcus aureus 25923. and Candida albicans 10231 were obtainedfrom American type culture collection (ATCC).

With regard to rr3ateri.als and equipment, silver(II) oxide was obtainedfrom Ames Goldsmith Inc. (New Jersey, USA). The silver(II) oxidetypically contains at least 90% Ag₄O₄, by weight, and may contain someAgO and Ag₂O.

Ag₂O (Catalog no. S1090), also known as silver(I) oxide, was obtainedfrom Spectrum Chemicals (New Jersey, USA).

4-phenylbutyric acid and 2-ethyl-hexanoic acid were obtained fromSigma-Aldrich (items P21005 and 538701, respectively).

The SYTO® bacterial count kit and LIVE/DEAD® Funga Light™ YeastViability Kit were obtained from Invitrogen Inc. (Texas, USA). Bacterialculture broths and media were obtained from R.emel Inc. (New York, USA).

The microbial shaking incubator (model 311DS Labnet Inc.) and AttuneFlow cytometer (Invitrogen Inc.) were used for bacterial culture andbacterial assay, respectively.

¹H and ¹³C-NMR spectra were obtained on a Balker DPX-300 spectrometer.Chemical shifts are expressed in ppm downfield from Me₄Si (TMS), whichis used as an internal standard. The values are given in the δ scale.

Low Resolution Mass Spectra (LRMS) were also obtained on a Q-TOF micro(Waters UK) spectrometer using ESI (Electron Spray Ionization).

Melting points were determined on a Fisher-johns apparatus and wereuncorrected.

Elemental silver analysis was carried out by Inductively-Coupled Plasma(ICP) Atomic Emission Spectroscopy (Ultima 2, Jobin Yvon Horiba).

Electron Spin Resonance (ESR) spectra were obtained on an X-band ElexsysE500 EPR spectrometer (Bruker, Karlsrube, Germany).

Example 1

An exemplary general procedure for producing the formulations (e.g.,ointments and silver carboxylate concentrates) according to the presentinvention is as follows:

The silver(II) oxide powder is weighed in a weighing dish. Thecarboxylic acid containing material, typically solid at roomtemperature, is weighed in the reaction vessel, and heated, typically toa temperature within the range of 88-9.3° C., to obtain a liquid medium.The black silver(II) oxide powder is introduced to the hot liquid, andthe reaction mixture is vigorously stirred. Stirring is continued whilemaintaining the temperature at 88-93° C. for the remainder of thisstage. Typically, the solution will gradually lighten as the reactionmixture is maintained at 88-93° C. A general color progression may beobserved: the color of the reaction mixture may turn from black (afteradding the silver(II) oxide) to olive green to dark yellow. In manycases, the color of the reaction mixture may continue to develop into alight yellow, and may further develop to off-white and finally to white.

The reaction time may be about 1.5 to 48 hours, depending on the natureof the particular carboxylate, the molar ratio of the silver(II) oxideto the carboxylic acid functional group, mixing conditions (includingviscosity), and temperature. Optionally, the reaction may bedeliberately curtailed in order to ensure a presence of Ag₄O₄ within theformulation.

For particularly viscous carboxylic acid containing materials, theviscosity of the reaction mixture may be lowered by introducing a lowercarboxylic acid (e.g., a C12 acid), by introducing a lower (low carbonnumber) silver carboxylate (e.g., a silver(II) C12 carboxylate), or byreturning a portion of the product material from a previously-producedbatch of the silver(II) carboxylate.

The product material from Example 1 may undergo further formulation. Forexample, the product material may be mixed with an oil and/or a liquidwax ester such as jojoba oil. Optionally, one or more silver(I)carboxylates may be blended into the formulation. Also, one or moreessential oils such as palmarosa oil may be introduced. Stirring may becontinued, typically for 0.5 to 2 hours, during cooling of the mixtureto below about 40° C. The formulation may then be poured into storagecontainers.

Example 2

An exemplary general procedure for producing an emulsion-basedsilver(II) carboxylate compositions and formulations according to thepresent invention is as follows: a liquid such as water may be mixed orblended at high speed in a formulation vessel, preferably with athickening agent such as bentonite or hectorite. Mixing may be continuedas the silver(II) carboxylate, preferably heated, is introduced.Optionally, an essential oil such as palmarosa oil may be added.

In some cases, a liquid wax ester may be heated and mixed with thesilver(II) carboxylate, prior to introduction of the carboxylate to theformulation vessel.

Higher concentrations of the thickening agent (e.g., 4-7% bentonite) maybe associated with thicker creams; lower concentrations of thethickening agent (e.g., 1-2% bentonite) may be associated with creamshaving a relatively low viscosity.

Example 3

30 grams of beeswax and/or a carboxylic acid is melted on a hot plate,typically at a temperature of approximately 100° C. After melting of thebeeswax and/or the fatty acid, 1 gram of AgO is added. Care should betaken to ensure that the mixture does not burn.

From the initial dark black color, the mixture typically lightens,ultimately turning light yellow, off-white, or white. 20 g ofemulsifying wax is then melted at around 90° C., and subsequently addedto the above-described reaction mixture, and stirred vigorously. Thetemperature may then be slowly lowered to about 75° C. Care should betaken not to lower the temperature too much as it ma cause hardening ofthe wax.

Separately, 680 grams of water and 70 grams of Bentonite clay may beblended in a high shear blender. The mixture may then be blended at thehigh setting for about 60 minutes.

Subsequently, 240 grams of jojoba oil may be added to the above mixture,while continuing with the blending process operation. At this point, 50grams of the Ag-beeswax/carboxylic acid-emulsifying mixture may be addedto the emulsion, while intensive blending may be continued for 40minutes. The end product may be an emulsion containing a silver(II)carboxylate.

Example 4

A mixture of Ag₄O₄ (0.3 grams, 24.22 mmol) and 4-phenylbutyric acid (anaromatic aliphatic carboxylic acid) (3.97 g, 2.42 mmol) was stirred at90° C. overnight. The reaction mixture, initially black, eventuallyturned white. A sample of about 10 mg of the white solid obtained wasmixed with water and a small amount of concentrated ammonia and an ESRspectrum of the solution was taken. The ESR spectrum obtained, providedin FIG. 1, indicates the presence of a free radical such as Ag⁺². Basedon various testing procedures described hereinbelow, the reactionproduct is silver(II) di-4-phenylbutyrate.

An ESR spectrum of Ag(II) picolinate is provided in FIG. 2 forcomparative purposes.

Example 5

A similar reaction to that described in Example 4 was carried out withAg₄O₄ (0.2 grams, 1.61 mmol), while replacing the 4-phenylbutyric acidwith 2-ethylhexanoic acid (1.86 grams, 10 mmol), a branched aliphaticcarboxylic acid. The reaction mixture, initially black, graduallylightened, and ultimately turned white. ¹³C-NMR (300 MHz, DMSO-D₆)preformed on the white solid yielded ppm δ in the 2-ethyl-hexanoic acid,the COOH carbon has a chemical shift of 177.34 ppm, whereas in theisolated silver salt the COO⁻ carbon has a chemical shift of 179.4 ppm.

Example 6

Three (3.0) grams of Ag₄O₄ were introduced to 40 grams of palmitic acid.The reaction mixture was maintained within a range of 90° C. to 110° C.overnight, resulting in a white waxy material.

The resulting material was sent for infra red (IR) spectroscopy analysisand the results were compared to the spectrum of palmitic acid used inthe synthesis. The IR plot obtained is provided in FIG. 3.

Peaks at wavenumbers 1519 and around 1420 (not resolved) may indicatethe COO— asymmetric and symmetric stretching vibrations of acarboxylate. The 1519 peak exactly matches the peak for silver behenatereported by Liu et al. in “Thermal Decomposition Process of SilverBehenate”, Thermochimica Acta 440 (2006) 1-6, Elsevier Press. Thesepeaks are absent from the palmitic acid sample.

In addition, the C═O stretch at wavenumber 1703 and the C—O stretch atwavenumber 1296 are reduced from the fatty acid to the product,suggesting that the number of COOH groups is diminishing.

These pieces of evidence strongly suggest the formation of a silvercarboxylate complex.

Examples 7-10

All of the reactions carried out in Examples 10 were performed using 1.5grams of Ag₄O₄, containing about 0.012 moles of silver. These reactionswere performed using n-Docosanol as a medium or vehicle, where theweight of the n-Docosanol and the fatty acid totaled 40 grams. Thepalmitic add used contained 97.4% palmitic acid; the discrepancy fromthe pure material was accounted for in the molar calculations.

The results are summarized in Table 1. For molar ratios of 2:1(carboxylic group Ag₄O₄, on an AgO basis) and above, a white solid wasobtained, whereas for a molar ratio of 1.6:1, a light brown solid wasobtained. It would appear than there was not enough carboxylic acid toreact with the Ag₄O₄, consequently, the remaining Ag₄O₄, reacted withthe solvent, or perhaps remained partially unreacted.

Additional support for the valence of the silver in the carboxylate isprovided hereinbelow.

TABLE 1 Palmitic Acid Ag₄O₄ Example moles moles Molar Ratio Final No. gCOO g Ag (Ag:COO) Color 7 20 0.076 1.5 0.012 6.3:1 White 8 10 0.038 1.50.012 3.2:1 White 9 6.4 0.024 1.5 0.012 2.0:1 White 10 5 0.019 1.5 0.0121.6:1 Light Brown

Examples 11-14

All of the reactions carried out in Examples 11-14 were performed using1.5 grams of Ag₄O₄, containing about 0.012 moles of silver. Thesereactions were performed using n-Docosanol as a medium or vehicle, wherethe weight of the n-Docosanol and the fatty acid totaled 40 grams. Thebehenic acid used contained 89.3% behenic acid; the discrepancy from thepure material was accounted for in the molar calculations.

The results are summarized in Table 2:

TABLE 2 Behenic Acid Ag₄O₄ Example moles moles Molar Ratio Final No. gCOO g Ag (Ag:COO) Color 11 14.2 0.036 1.5 0.012 3.0:1 White 12 10.0~0.024 1.5 0.012 2.0:1 White (slightly off) 13 9.7 ~0.024 1.5 0.0122.0:1 Light Brown 14 5 0.019 1.5 0.012 1.0:1 Brown-Gray

For molar ratios above 2:1, a white solid was obtained. In Example 13,slightly less behenic acid (0.3 grams less) was reacted with the Ag₄O₄,and the reaction mixture turned a light brown. As above, it would appearthan there was not enough carboxylic acid to react with the Ag₄O₄,consequently, the excess Ag₄O₄, reacted with the solvent, or perhapsremained partially unreacted.

At a molar ratio of 1:1, significantly below 2:1, the reaction mixtureturned brown-gray.

Examples 15-17

Each of the silver(II) palmitate of Example 7, silver(II) behenate ofExample 11, and silver(II) 4-phenylbutyrate of Example 4 wereincorporated into a carrier base containing beeswax and jojoba oil toproduce the formulations of Examples 15-17 (1%/20% beeswax/79% jojobaoil). The formulation exhibited physical and chemical stability. Each ofthe silver(II) palmitate (Example 15), silver(II) behenate (Example 16),and silver(II) 4-phenylbutyrate formulations was examined over thecourse of 3-12 months to assess long term physical and chemicalstability. No degradation of the formulations was observed: there wassubstantially no physical segregation of phases, and the formulationsretained their light/white color over time.

Other samples of silver(II) carboxylates are the subject of an ongoingaging study. Various silver(II) carboxylate formulations have beensubjected to ultraviolet (UV) light in an accelerated test procedure.The results, along with various comparative examples, are provided belowin Examples 84-104.

Examples 18-29

A series of reactions between Ag₄O₄ and various aliphatic carboxylicacids w as conducted generally according to the synthesis procedureprovided in Example 1. In each synthesis, 80 grams of a particularcarboxylic acid (ranging from C8 to C26) or 80 grams of beeswax weretransferred into a reaction vessel.

All of these aliphatic carboxylic acids have melting points below 90° C.After melting the particular carboxylic acid or acids, as necessary, thetemperature may be slowly increased to about 90-100° C. The reaction maybe appreciably slower, or may fail to occur at lower temperatures, andabove about 105° C., various side reactions may occur, or evenpredominate.

Subsequently, 3 grams of Ag₄O₄ were introduced, and the reaction mixturewas stirred over the course of the reaction. A general color progressionwas observed the color of the reaction mixture typically turned fromblack (after adding the black silver(II) oxide powder) to olive green todark yellow. In many cases, the color of the reaction mixture continuedto develop, first into a light yellow, and with additional reactiontime, to off-white and finally to white.

The reactions generally appeared to go to completion within about 3-30hours, depending on the particular fatty acid.

About 320 grams of jojoba oil were heated to about 85° C. and wereintroduced to the reaction mixture after the reaction appeared complete.The mixture was then homogenized for about 30 seconds, gradually cooledto about 40° C., and transferred into storage containers.

The results of the reactions are summarized in Table 3 below.

Example 30

-   1. The bacterial cells were initially seeded in a culture flask    containing tryptic soy agar, as per ATCC guidelines, in a 311DS    incubator.-   2. The cells take approximately 30 hours to reach 90% confluence.    The cells were then individually seeded in test tubes containing 10    ml broth, and care was taken to maintain an identical cell count in    each tube.-   3. For each experimental time point, a control tube was seeded as    well.-   4. Sterile paper discs were handled in a biological hood and 1% of    the test formulation was carefully smeared therein.-   5. The disc was then dropped into the test tube with the bacterial    broth and placed into the shaking incubator. At the same time, an    “empty” disk was dropped into the control bacterial broth.-   6. The process was repeated several times, using various    formulations and run lengths.

Example 31

-   1. After the bacteria were allowed to be treated with a particular    formulation for the designated time point (20-180 min), the tubes,    along with a corresponding control, were removed from the incubator.    Using sterile tweezers, the disc was removed and discarded and the    bacteria were centrifuged at 5000 g for 30 seconds.-   2. The bacterial pellet was re-suspended in 2 mL of fresh tryptic    soy broth.-   3. At this point, 2 μL of the SYTO® bacterial stain (component A)    was added to the cells and incubated for 5 min.

TABLE 3 % Weight of Ag(II) Carbon MW Conversion Color after Stainingafter Moles of Acid:Ag(II) Carboxylate in Example Carboxylic Acid No.(g) Time (h) conversion UV exposure Acid molar ratio Formulation Example18 Caproic acid C6  116.2 NA Black NA 0.688 28.4 9.9% Example 19Caprylic acid C8  144.2 NA Brown NA 0.555 22.9 11.6% Example 20 Capricacid C10 172.3 4 h Light Gray Gray 0.464 19.2 13.2% Example 21 Lauricacid C12 200.3 3 h White Brown 0.399 16.5 14.9% Example 22 Palmitic acidC16 256.4 2 h White off-yellow 0.312 12.9 18.1% Example 23 Beeswax acids~C18  2-12 h White/very white off-white Example 24 Stearic acid C18284.4 3-4 h Very white White 0.281 11.6 19.8% Example 25 Nonadecyclicacid C19 298.5 5-6 h White Off-white 0.268 11.1 20.6% Example 26Arachidic C20 312.5 5-6 h Off white Off white 0.256 10.6 21.4% Example27 Behenic acid C22 340.5 8 h White Brown 0.235 9.7 23.1% Example 28Lignoceric acid C24 368.6 17 h Yellow Gray 0.217 9.0 24.7% Example 29Cerotic acid C26 396.7 29 h Dark yellow Gray 0.202 8.3 26.4%

-   4. After the incubation, 10 μL of component B were added, and    incubation was continued for another 5 minutes. The samples were    then analyzed using a flow cytometer. The flow cytometer provides    the results as a percentage of the control, and using that as a    scale, the number of cells may be mathematically determined.-   5. The bacterial count is ascertained based on the stainability with    the SYTO® stain. A new kit was used every 2 weeks, as the components    start degrading after about 15 days.

Using the above-mentioned technique, samples were analyzed for cellviability, and the results were documented, as provided hereinbelow.

Examples 32-41 Anti-Bacterial Performance of Aliphatic Silver(II)Carboxylates—S. Aureus

Using the above-provided techniques, the anti-bacterial performance ofmost of the aliphatic silver(II) carboxylates of Examples 18-29 wasevaluated, using Staphylococcus aureus 25923. The results over time areshown in a bar graph in FIG. 4.

Over the course of 4 hours of measurements, all 10 of thesilver(II)-containing formulations exhibited anti-bacterial activity. Ofthe 10 formulations, those having a carbon number above C12 appeared tobe particularly efficacious, such that after 4 hours, the microbialcount was reduced to about ⅕ to ⅓ of the control value.

Examples 42-51

Using the above-provided techniques, the anti-bacterial performance ofmost of the aliphatic silver(II) carboxylates of Examples 18-29 wasevaluated, using Escherichia coli 35218. The results over time are shownin a bar graph in FIG. 5.

Over the course of 4 hours of measurements, all 10 of thesilver(II)-containing formulations exhibited anti-bacterial activity. Ofthe 10 formulations, those having a carbon number above C12 appeared tobe particularly efficacious, such that after 4 hours, the microbialcount was reduced to about 0 to ⅓ of the control value

Example 52

-   1. After treating the fungus sample with a particular formulation    for the designated time point (20-180 min), the tubes, along with a    corresponding control, were removed from the incubator. Using    sterile tweezers, the disc was removed and discarded and the fungus    sample was centrifuged at 1000 g for 60 seconds.-   2. The fungal pellet was re-suspended in 2 ml of fresh saboraud    liquid media.-   3. At this point, 10 μl of the LIVE-DEAD® Funga Light™ Yeast    Viability Kit were added to the cells and incubated for 5 minutes-   4. The samples were then analyzed using a flow cytometer. The flow    cytometer provides the results as a percentage of the control, and    using that as a scale, the number of cells may be mathematically    determined.-   5. The fungal count is ascertained based on the stainability with    the LIVE/DEAD® Funga Light™ Yeast Viability Kit. A new kit was used    every 2 weeks, as the components start degrading after about 15    days.

Examples 53-62

Using the above-provided techniques, the anti-fungal performance of mostof the aliphatic silver(II) carboxylates of Examples 18-29 wasevaluated, using Candida albicans 10231. The results over time are shownin a bar graph in FIG. 6.

Over the course of 4 hours of measurements, all 10 of thesilver(II)-containing formulations exhibited anti-fungal activity. Ofthe 10 formulations, those having a carbon number above C12 appeared tobe particularly efficacious, such that after 6 hours, the microbialcount was generally reduced by more than ½ and more typically, by about60-75% relative to the control value.

Example 63

The synthesis procedure was conducted generally according to theprocedure provided in Example 1. 30 grams of palmitic acid were heatedin a reaction vessel to about 98° C.

Subsequently, 3 grams of Ag₄O₄ were introduced, and the reaction mixturewas stirred over the course of about 48 hours. The color of the reactionmixture, initially black from the black silver(II) oxide powder,eventually turned white.

The concentrated silver(II) palmitate (also known as silver(II)dipalmitate) produced had a calculated concentration of about 46%, theremainder consisting primarily of excess palmitic acid.

Example 64

The emulsion was prepared generally according to the procedure providedin Example 3. About 680 grams of water were introduced to a high shearblender, and 70 grams of Bentonite clay were added thereto. The mixturewas blended at a high setting for 50 minutes.

In a beaker, 240 grams of jojoba oil were heated to about 93° C., and 10grams of the concentrated silver(II) palmitate formulation of Example 62were introduced. The mixture was then homogenized for 1 minute.

This mixture was then added to the water-Bentonite base, and the newmixture was stirred at high speed for about 40 minutes.

The obtained formulation was an emulsion containing approximately 0.5%silver(II) palmitate.

Example 65

The emulsion was prepared according to the procedure provided in Example64, but only 2 grams of the concentrated silver(II) palmitateformulation of Example 63 were introduced.

The obtained formulation was an emulsion containing approximately 0.1%silver(II) palmitate.

Examples 66-68

1.4 grams Ag₄O₄ were introduced to heated beeswax, mixing to produce thesilver(II) carboxylate, using the process described in Example 1.

50 grams of the melted silver(II) carboxylate in beeswax were then addedto 50 grams of previously melted petrolatum (Example 66), AC-629(Example 6) and coconut oil (Example 68), in separate vessels, heated toabout 80° C. Each mixture was then homogenized and cooled.

All three of the ointments were found to act as powerful anti-microbialagents.

Examples 69-71

Ag₄O₄ was introduced to heated beeswax, as described in the previousexamples.

10 grams of the melted silver(II) carboxylate in beeswax were then addedto 50 grams of previously melted petrolatum (Example 69), AC-629(Example 70) and coconut oil (Example 71), in separate vessels, heatedto about 80° C. Each mixture was then homogenized and cooled.

All three of the ointments were found to act as powerful anti-microbialagents.

Example 72

An ointment based on silver(I) carboxylate and jojoba oil was preparedgenerally according to Example 1, but using a commercially-availablesilver(I) palmitate.

The ointment, containing about 2.2% silver carboxylate, by weight, wasfound to exhibit anti-microbial activity.

Example 73

An ointment based on silver(I) carboxylate was prepared generallyaccording to Example 66, but using a commercially-available silver(I)stearate, instead of reacting the silver(II) oxide with beeswax.

The ointment, containing about 1.3% silver carboxylate, by weight, wasfound to exhibit anti-microbial activity.

Example 74

Silver(II) palmitate from Example 22 and silver(I) palmitate fromExample 72 were heated separately and mixed in a 1:6 ratio to produce amixed silver(I)-silver(II) Carboxylate Formulation. The resultingformulation was found to exhibit anti-microbial activity.

Example 75

Silver(II) laurate from Example 21 and silver(I) palmitate from Example72 were heated separately and mixed in a 1:9 ratio. The resultingformulation was found to exhibit anti-microbial activity.

Example 76

Determination of the organic acid bound to the silver in a cream orointment, and determination of the oxidation state of the silver ion inthe silver carboxylate found therein, may be performed as follows:

A sample of the cream was mixed with toluene to remove lipophilicmaterials, leaving organic salts in the solid or semi-solid phase. Themixture was centrifuged and the supernatant was decanted. The residuewas washed with ether to remove remaining traces of toluene, and theresidue was dried.

The residue was then mixed with an excess of trifluoroacetic acid, toconvert any silver carboxylates to silver trifluoroacetate, and freeingthe carboxylates as the corresponding free carboxylic acids. Theobtained mixture was evaporated. The residue was mixed with ether andcentrifuged, and the supernatant was separated and evaporated.

The residue, obtained after evaporation of the ether, was analyzed by¹H-NMR (300 MHz, CDCl₃). The residue produced a spectrum having thefollowing characteristics: ppm δ 0.90 (t, 3H), 1.25 (bs, 28H), 1.61 (q,2H), 2.24 (t, 2H), which indicates that the compound present was stearicacid. However, the integration of the peak at 1.25 ppm indicates thepresence were a small amount of additional hydrogens, which may beattributable of a small amount (less than 10% of the acids present) ofbehenic acid.

A second sample of the residue (207 mg) obtained after mixing the creamwith toluene, was dissolved in a small amount of concentrated nitricacid. The nitric acid solution was washed with ether, the ether wasdecanted, and the aqueous phase was diluted with water.

An elemental silver analysis by ICP revealed that the amount of thesilver obtained (33.6 mg/L) corresponded, within a ˜97% accuracy, to thecalculated concentration of silver in a mixture of silver distearate(90%) and silver dibehenate (10%).

The 207 mg of residue, when composed of 90/10 distearate/dibehenate,gives a calculated value of silver of 32.5 mg/L. This theoretical valueof 32.5 mg/L is within 3% of the found value of 33.6 mg/L, indicatingthat the oxidation state of the silver ion was Ag⁺². Had the saltobtained been that of Ag⁺¹ ion, the calculated amount of silver in 207mg of residue would have been 49 mg/L.

Moreover, the ESR spectrum obtained, provided in FIG. 7, would appear toconfirm the presence of Ag⁺².

Example 77

Formulation reflectance, lightness, or whiteness was evaluated asfollows: approximately 1 gram of a particular sample (typically anointment or cream) was spread on a 5 cm by 5 cm area of white cottoncloth and distributed evenly, typically using a metal spatula.

A LabScan XE spectrophotometer instrument (HunterLab, VA) was used toevaluate the reflectance of each sample. The working principle of theinstrument pertains to the property of light reflection. The clothsample is stored in a completely dark container. To measure thereflectance, the instrument exposes the sample to a controlled,repeatable pulse of light. The lightness of the sample is generallycorrelated with the reflectance: higher values correspond to lightersamples.

The spectrophotometer has a wavelength range of 375 nm to 750 nm and anoptical resolution of 10 nm. The spectrophotometer measures reflectedcolor using a 0°/45° geometry.

Example 78

Formulation reflectance was evaluated as a function of the exposure timeto ultraviolet light, as follows: the LabScan XE spectrophotometerdescribed in Example 74 was used. Each sample was continuously exposedto ultraviolet light produced by the illumination source. The continuousUV exposure is through a 254 nm, 6 W UV bulb distributed byCole-Parmer®. The distance between the UV source and the specimen orformulation was 18 inches (˜45.7 cm).

Sample preparation was substantially the same as that described inExample 74. After an initial measurement (“time 0”), additionalmeasurements were made over the course of the exposure to ultravioletlight, typically after 12 or 24 hours.

Examples 79-83

FIG. 8 is a bar graph plotting formulation whiteness for formulationscontaining: (1): Ag₄O₄ (0.7%); (2): Ag₂O (0.7%); and (4): Ag(II)picolinate (0.7%), each in a base containing beeswax (19.8%) and jojobaoil (79.5%), vs. an inventive formulation containing 0.7% silverdistearate in a substantially identical base.

Examples 84-87

FIG. 9 is a bar graph plotting formulation whiteness for prior-artformulations containing: (1): Ag₄O₄ (0.1%); (2): Ag₄O₄ (0.7%); (3):Ag₄O₄ (1.5%); and (4): Ag₄O₄ (3%), each in a base containing jojoba oiland beeswax in about a 4:1 weight ratio. After an initial measurementprior to UV exposure, an additional measurement was made after 12 hoursof exposure to ultraviolet light (see Examples 77-78).

Examples 88-91

FIG. 10 is a bar graph plotting formulation whiteness for prior-artformulations containing: (1): Ag₂O (0.1%); (2): Ag₂O (0.7%); (3): Ag₂O(1.5%); and (4): Ag₂O (3%), each in a base containing jojoba oil andbeeswax in about a 4:1 weight ratio. After an initial measurement priorto UV exposure, an additional measurement was made after 12 hours ofexposure to ultraviolet light (see Examples 77-78).

Examples 92-95

FIG. 11 is a bar graph plotting formulation whiteness for formulationsaccording to the present invention, the formulations containing: (1):Ag(II) carboxylate (0.1% on an Ag₄O₄ basis); (2): Ag(II) carboxylate(0.7% on an Ag₄O₄ basis); (3): Ag(II) carboxylate (1.5% on an Ag₄O₄basis); and (4): Ag(II) carboxylate (3% on an Ag₄O₄ basis); eachdisposed in a base containing jojoba oil and beeswax in about a 4:1weight ratio. The Ag(II) carboxylate was produced from Ag₄O₄ andbeeswax. After an initial measurement prior to UV exposure, anadditional measurement was made after 12 hours of exposure toultraviolet light (see Examples 77-78).

Examples 96-98

FIG. 12 is a bar graph plotting formulation whiteness for prior-artformulations containing: (1): 5% ZnO and 0.7% Ag₄O₄; (2): 5% MgO and0.7% Ag₄O₄; and (3): 5% TiO₂ and 0.7% Ag₄O₄, each in a base containingjojoba oil and beeswax in about a 4:1 weight ratio. After an initialmeasurement prior to UV exposure, an additional measurement was madeafter 12 hours of exposure to ultraviolet light (see Examples 77-78).

Examples 99-101

FIG. 13 is a bar graph plotting Formulation whiteness for formulationsaccording to the present invention, the formulations containing (1):Ag(II) carboxyl ate (0.7% on an Ag₄O₄ basis) and 5% ZnO; (2): Ag(II)carboxylate (0.7% on an Ag₄O₄ basis) and 5% MgO; and (3): Ag(II)carboxylate (0.7% on an Ag₄O₄ basis) and 5% TiO₂; each disposed in abase containing jojoba oil and beeswax in about a 4.1 weight ratio. TheAg(II) carboxylate was produced from Ag₄O₄ and beeswax After an initialmeasurement prior to UV exposure, an additional measurement was madeafter 12 hours of exposure to ultraviolet light (see Examples 77-78).

Examples 102-104

FIG. 14 is a bar graph plotting formulation whiteness for formulationsaccording to the present invention, the formulations containing: (1):Ag(II) carboxylate (0.7% on an Ag₄O₄ basis) in a petrolatum base; (2):Ag(II) carboxylate (0.7% on an Ag₄O₄ basis) in an AC-629 base; (3):Ag(II) carboxylate (0.7% on an Ag₄O₄ basis) in a coconut oil base. TheAg(II) carboxylate was produced from Ag₄O₄ and beeswax. After an initialmeasurement prior to UV exposure, an additional measurement was madeafter 12 hours of exposure to ultraviolet light (see Examples 77-78).

Examples 105-113

Ag(II) carboxylate formulations were prepared according to the procedureprovided for Examples 21-29, but with 2.4 grams of silver(II) oxide(instead of 3 grams). After being stored in airtight plastic storagecontainers, the color and texture of the formulations were monitored intri-monthly fashion. These formulations exhibited no phase separation ordarkening/color change after three months, six months, 9 months or 1year of storage at room temperature. To date, after 14 months ofstorage, the formulations look substantially identical to similar, newlyproduced Ag(II) carboxylate formulations. Moreover, the aged formulationcontinued to exhibit strong anti-microbial activity.

Example 114 Viral Assay Protocol Seeding Healthy NRK 52E (Rat KidneyEpithelial Cells):

1. Make three substantially identical base solutions of cells, eachsolution having 1·10⁶ cells per mL in Growth Media:

Solution 1: control solution;

Solution 2: introduce to the second base solution about 0.1% by weightof an inventive homeopathic, wart formulation containing 0.7% silver(II)carboxylate on an AgO basis, 0.2% liquid containing alcohol (67%), water(33%), and a trace of thuja occidental extract, all disposed within abase containing ˜3.5:1 jojoba oil to beeswax; and

Solution 3: introduce to the third base solution about 0.1% by weight ofan inventive wart stick containing 0.7% silver(II) carboxylate on an AgObasis and salicylic acid (12%) both disposed within a base containing˜5:1 jojoba oil to beeswax;

2. Vortex all three solutions;

3. Add 1.0 mL of growth media with cells to a 6 well plate; and

4. Incubate plates in a 37° C., 5% CO₂ incubator. After ˜12-24 hours,the cells should be ˜90-100% confluent.

Preparing Adenovirus for Infection:

1. Prepare 4 tubes, each tube containing 2 mL of PBS;

2. Add 20 μL of virus sample to the first tube;

3. Vortex thoroughly.

Infecting the Cells with the Virus

1. Pipette off and discard 5 mL of media from each well. One mL of mediashould now remain in each monolayer;

2. Add 100 μL of the viral solution to each well; at this point thereshould be three 6 well plates: control, terrasil with homeopathic andterrasil with salicylic acid, each infected with adenovirus;

3. Incubate the infected monolayer/s for 24 hours at 37° C.

Agar Overlay:

1. Prepare a sterile solution of 4% agarose in distilled water byautoclaving at 121° C. for 20 minutes;

2. Cool down the agarose to 37° C.;

3. Gently overlay 3 mL of agarose into each well of the three samples,and allow to solidify for 15 minutes;

4. Move the plate(s) to a humidified incubator at 37° C. having 10% CO₂;

Deep purple plaques will be visible 48-72 hours after infection. Plaquesare visible to the naked eye and can be counted by placing the wellplates on top of a light source.

Examples 115-116

Viral Assay - Results Approximate No. Sample No. of Plaques Control 67Example 114: 21 Example 115: 13

Example 117

Reaction between Ag₄O₄ and linoleic acid (a polyunsaturated omega-6fatty acid) was conducted generally according to the synthesis procedureprovided in Example 1. Linoleic acid, a liquid at room temperature, washeated to ˜93° C. in a reaction vessel.

Subsequently, 3.75 grams of Ag₄O₄ were introduced, and the reactionmixture was stirred over the course of the reaction. The color of thereaction mixture gradually turned from dark gray, after adding the blacksilver(II) oxide powder, to yellow to off-white. The white solidsproduced were separable from the liquor, and displayed anti-microbialefficacy.

Example 118

Reaction between Ag₄O₄ and oleic acid (a monounsaturated fatty acid) wasconducted according to the synthesis procedure provided in Example 117,using the same quantities of Ag₄O₄ and fatty acid. The color of thereaction mixture gradually turned from dark gray, after adding the blacksilver(II) oxide powder, to yellow to off-white. The white solidsproduced were separable from the liquor, and displayed anti-microbialefficacy. The conversion of linoleic acid appeared to transpire morerapidly than the conversion of oleic acid.

The inventors believe that in the formulations of the present invention,ketone moieties may substitute hydrogen moieties along the length of thehydrocarbon chain (except within 1-2 positions from the acid moiety).Such structures may be largely impervious attach by the silver(II) oxideduring the synthesis. Similarly, halogen moieties, most typicallychlorine moieties, may generally substitute hydrogen moieties along thelength of the hydrocarbon chain.

The methods provided herein were found to be particularly suitable forcarboxylic acids, and more specifically, aliphatic carboxylic acids suchas fatty acids, having a melting point below 105° C., and moretypically, below 102° C., below 100° C., below 98° C., below 96° C.,below 93° C., or below 90° C. Carboxylic acids having melting pointswell below these temperatures tended to convert to the (Ag⁺²)carboxylate with yet greater facility.

The conversion of monounsaturated and polyunsaturated acids may occurwith relative facility with respect to the corresponding saturated acid.

As used herein in the specification and in the claims section thatfollows, the term “antimicrobial”, with respect to a chemical agent orformulation, refers to a substance that is destructive tomicroorganisms, or inhibits the growth thereof.

As used herein in the specification and in the claims section thatfollows, the term “antibiotic” refers to a substance that selectivelyattacks and destroys at least one species or type of microorganism,while exhibiting relative inertness with respect to human and/ormammalian cells. More typically, the antibiotic substance selectivelyattacks and destroys at least one species or type of microorganism thatcommonly populates the skin, surface wounds, bedsores and the like,while exhibiting relative inertness, with respect to skin cells ofhumans and/or mammals. The term “antibiotic” is specifically meant toexclude anti-microbial preservatives, both anti-fungal preservatives andanti-bacterial preservatives. Such anti-fungal preservatives include,but are not limited to, compounds such as benzoic and ascorbic acids andalkali salts thereof, and phenolic compounds such as methyl, ethyl,propyl and butyl p-hydroxybenzoate (parabens). Antibacterialpreservatives include, but are not limited to, compounds such asquaternary ammonium salts, alcohols, phenols, mercurials andbiguanidines. The term “antibiotic” is specifically meant to excludeanti-microbial preservatives such as table salt and the like, vinegar,sodium nitrate, sodium nitrite, and sulfites. The term “antibiotic” isspecifically meant to include, without being limited to, silver oxidessuch as silver(I) oxide and silver(II) oxide, silver sulfadiazine, andany other topical antibiotics that are efficacious in the treatment ofserious skin wounds such as bedsores, skin ulcers, and puncture wounds,or that are efficacious in the treatment of mundane skin wounds. Theterm “antibiotic” is specifically meant to include “classic” topicalantibiotics such as Bacitracin, Neomycin, Erythromycin andChloramphenicol. Additional topical antibiotic substances may be readilyapparent to those of ordinary skill in the art.

As used herein in the specification and in the claims section thatfollows, the term “therapeutically effective amount”, with respect to anantibiotic substance or formulation, refers to a quantity sufficient toproduce a positive result in the treatment of at least one topicalinfection.

As used herein in the specification and in the claims section thatfollows, the term “therapeutically effective concentration”, withrespect to an antibiotic substance within a formulation or medicaldevice, refers to a concentration of the antibiotic, within theformulation or medical device, sufficient to produce a positive resultin the treatment of at least one topical infection.

As used herein in the specification and in the claims section thatfollows, the term “percent”, or “%”, refers to percent by weight, unlessspecifically indicated otherwise.

As used herein in the specification and in the claims section thatfollows, the term “Ag₄O₄ weight basis” refers to a weight basis obtainedby multiplying the silver weight content of a material by a factor of(AgO/Ag), or about 1.148.

Similarly, the term “ratio”, as used herein in the specification and inthe claims section that follows, refers to a weight ratio, unlessspecifically indicated otherwise.

As used herein in the specification and in the claims section thatfollows, the term “silver(II) oxide” refers to a silver oxide whose unitstructure contains silver and oxygen in a substantially 1:1 molar ratio.The term “silver(II) oxide” is specifically meant to include Ag₄O₄(often represented as Ag₂O₃.Ag₂O) and AgO.

As used herein in the specification and in the claims section thatfollows, the term “nominal valence”, with respect to silver in a silvercarboxylate, refers to an average valence of the silver within thesilver carboxylate compound (molecule, salt, complex, etc.). Thus, byway of example, silver in a silver(II) carboxylate represented asAg(OOCR)₃.AgOOCR would have a nominal valence of 2.

As used herein in the specification and in the claims section thatfollows, the term “corresponding” with respect to a carboxylic acid,refers to the acid form of a silver carboxylate. Thus, palmitic acid isthe corresponding carboxylic acid of a silver palmitate.

As used herein in the specification and in the claims section thatfollows, the term “silver containing compound” is specifically meant toexclude metallic silver (Ag⁰). The term “silver containing compound” isspecifically meant to include dissociated silver species, and/or silverspecies forming a complex.

As used herein in the specification and in the claims section thatfollows, the term “silver(I) oxide” refers to a silver oxide whose unitstructure contains silver and oxygen in a substantially 2:1 molar ratio.The term “silver(I) oxide” is specifically meant to include Ag₂O.

As used herein in the specification and in the claims section thatfollows, the term “standard whiteness value”, and the like, is meant torefer to the procedure detailed in Example 77.

As used herein in the specification and in the claims section thatfollows, the term “standard ultraviolet light (UV) treatment”, and thelike, is meant to refer to the procedure detailed in Example 78.

As used herein in the specification and in the claims section thatfollows, the term “largely includes”, “consists largely of” and thelike, with respect to a component within a formulation, refers to acontent of at least 30%, by weight; the term “mainly includes”,“consists mainly of,” and the like, refers to a content of at least 50%,by weight; the term “predominantly includes”, “consists predominantlyof”, and the like, refers to a content of at least 65%, by weight.

It will be appreciated that certain features of the invention, whichare, for clarity, described in the context of separate embodiments, mayalso be provided in combination in a single embodiment. Conversely,various features of the invention, which are, for brevity, described inthe context of a single embodiment, may also be provided separately orin any suitable sub-combination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

The following statements were recited in the parent case as claims:

-   A. An antimicrobial formulation comprising:    -   (a) at least one silver-containing compound, including an        anti-microbial agent containing an aliphatic silver carboxylate,        said silver of said aliphatic carboxylate having a nominal        valence of 2, said at least one silver-containing compound        having an average valence of at least 1.1; and    -   (b) a carrier base;    -   said at least one silver-containing compound being dispersed        within said base.-   B. The formulation of statement A, the formulation having a form of    a cream, an emulsion, or an ointment.-   C. The formulation of statement A or B, a total silver content of    the formulation, or a total silver content of said at least one    silver-containing compound, being within a range of 0.0005% to 20%,    0.0005% to 17%, 0.0005% to 7%, 0.0005% to 3.5%, 0.0005% to 3%,    0.0005% to 2.5%, 0.001% to 3.5%, 0.005% to 3.5%, 0.01% to 3.5%,    0.03% to 3.5%, 0.05% to 3.5%, 0.10% to 3.5%, 0.30% to 3.5%, 0.5% to    3.5%, 0.7% to 3.5%, or 0.9% to 3.5%, by weight.-   D. The formulation of any one of statements A to C, a content of    said aliphatic carboxylate being at least 0.1%, on an Ag₄O₄ weight    basis, the formulation being white or at least off-white.-   E. The formulation of any one of statements A to C, a content of    said aliphatic carboxylate being within a range of 0.1% to 1.7%, on    an Ag₄O₄ weight basis, the formulation having a standard whiteness    value of at least 3.4, at least 3.5, at least 3.6, at least 3.7, at    least 3.8, or at least 3.9 reflective units (RU).-   F. The formulation of statement C, said total silver content of said    at least one silver-containing compound being within a range of    0.09% to 1.7%, on an Ag₄O₄ weight basis, the formulation having a    standard whiteness value of at least 4.0, at least 4.1, at least    4.2, or at least 4.3 reflective units (RU).-   G. The formulation of statement C, said total silver content of said    at least one silver-containing compound being within a range of 0.8%    to 3.4%, by weight, the formulation having a standard whiteness    value of at least 3.5, at least 3.6, at least 3.7, at least 3.8, or    at least 3.9 reflective units (RU).-   H. The formulation of statement E or F, at least one of said    aliphatic silver carboxylate and said carrier base selected, such    that after standard ultraviolet light (UV) treatment, in which the    formulation is subjected to constant exposure to UV for 12 hours at    240 nm, said standard whiteness value of the formulation remains    within 0.6 RU, within 0.5 RU, within 0.4 RU, within 0.3 or within    0.2 RU, of an initial whiteness value of the formulation prior to    said treatment.-   I. The formulation of any one of statements A to H, at least one of    said silver carboxylate and said carrier base selected, such that    after standard ultraviolet light (UV) treatment, in which the    formulation is subjected to constant exposure to UV for 12 hours at    240 nm, a post-UV whiteness value of the formulation remains at    least 3.5 reflective units (RU), at least 3.6 RU, at least 3.7 RU,    at least 3.8 RU, at least 3.9 RU, at least 4.0 RU, or at least 4.1    RU.-   J. The formulation of statement H or I, the formulation containing    said silver carboxylate in a range of 0.30% to 3.5%, 0.4% to 3.5%,    0.5% to 3.5%, 0.6% to 3.5%, 0.7% to 3.5%, 0.30% to 3%, 0.4% to 3%,    0.5% to 3%, or 0.6% to 3%, by weight.-   K. The formulation of any one of statements A to J, said average    valence being at least 1.2, at least 1.3, at least 1.4, at least    1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, or    about 2.0.-   L. The formulation of any one of statements A to K, the formulation    containing less than 5%, less than 4%, less than 3%, less than 2.5%,    less than 2.0%, less than 1.5%, less than 1.2%, less than 1%, less    than 0.8%, less than 0.6%, or less than 0.4% of a whitening agent.-   M. The formulation of any one of statements A to L, the formulation    containing less than 10%, less than 9%, less than 8%, less than 6%,    less than 4%, less than 2%, less than 1%, less than 0.5%, less than    0.2%, less than 0.1%, or less than 0.05% of silver-containing    compounds selected from the group consisting of silver(II) fluoride    (AgF₂), silver(II) picolinate (C₁₂H₈AgN₂O₄), silver(I) oxide and    silver(II) oxide.-   N. The formulation of any one of statements A to M, said at least    one silver carboxylate including an at least divalent silver    carboxylate having a carbon number or average carbon number less    than 40, less than 38, less than 36, less than 34, less than 33,    less than 32, less than 31, less than 30, less than 29, less than    28, less than 27, less than 25, less than 23, less than 22, or less    than 21.-   O. The formulation of any one of statements A to N. said at least    one silver carboxylate including an at least diwilent silver    carboxylate having a carbon number or average carbon number greater    than 9, greater than 10, or greater than 11.-   P. The formulation of any one of statements A to O, the formulation    containing at least one carboxylic acid, said at least one    carboxylic acid optionally including a corresponding carboxylic acid    of said silver carboxylate having said nominal valence of 2, a molar    ratio of said corresponding carboxylic acid to said silver    carboxylate having said nominal valence of at least 2 optionally    being at least 0.01, at least 0.025, at least 0.05, at least 0.1, at    least 0.2, at least 0.5, at least 1.0, at least 1.5, at least 2, or    at least 3.-   Q. The formulation of any one of statements A to P, a hydrocarbon    structure of said carboxyl ate selected from the group consisting of    fully saturated, monounsaturated, and polyunsaturated structures.-   R. The formulation of any one of statements A to Q, a backbone    structure of said carboxylate including a structure selected from    the group consisting of a straight hydrocarbon chain and a branched    hydrocarbon chain.-   S. The formulation of any one of statements A to R, a backbone    structure of said carboxylate including a structure selected from    the group consisting of a ring structure and an aromatic structure.-   T. The formulation of statement I, the formulation having a form of    a cream, an emulsion, or an ointment, a total silver content of the    formulation being within a range of 0.0005% to 20%, said aliphatic    silver carboxylate including a silver carboxylate of a fatty acid.

1. A method of producing a silver(II) carboxylate or silver(II)carboxylate formulation, the method including the steps of: (a) mixing acarboxylic acid and silver(II) oxide to produce a reaction mixture; and(b) heating the reaction mixture to produce an aliphatic silvercarboxylate, the silver of the aliphatic silver carboxylate having anominal valence of
 2. 2. An antimicrobial formulation comprising: (a) atleast one silver-containing compound, including an anti-microbial agentcontaining an aliphatic silver carboxylate, said silver of saidaliphatic carboxylate having a nominal valence of 2, said at least onesilver-containing compound having an average valence of at least 1.1;and (b) a carrier base; said at least one silver-containing compoundbeing dispersed within said base.
 3. The formulation of claim 2, theformulation having a form of a cream, an emulsion, or an ointment. 4.The formulation of claim 2, a total silver content of the formulation,or a total silver content of said at least one silver-containingcompound, being within a range of 0.0005% to 20%, 0.0005% to 12%,0.0005% to 7%, 0.0005% to 3.5%, 0.0005% to 3%, 0.0005% to 2.5%, 0.001%to 3.5%, 0.005% to 3.5%, 0.01% to 3.5%, 0.03% to 3.5%, 0.05% to 3.5%,0.10% to 3.5%, 0.30% to 3.5%, 0.5% to 3.5%, 0.7% to 3.5%, or 0.9% to3.5%, by weight.
 5. The formulation of claim 2, a content of saidaliphatic carboxylate being at least 0.1%, on an Ag₄O₄ weight basis, theformulation being white or at least off-white.
 6. The formulation ofclaim 2, a content of said aliphatic carboxylate being within a range of0.1% to 1.7%, on an Ag₄O₄ weight basis, the formulation having astandard whiteness value of at least 3.4, at least 3.5, at least 3.6, atleast 3.7, at least 3.8, or at least 3.9 reflective units (RU).
 7. Theformulation of claim 4, said total silver content of said at least onesilver-containing compound being within a range of 0.09% to 1.7%, on anAg₄O₄ weight basis, the formulation having a standard whiteness value ofat least 4.0, at least 4.1, at least 4.2, or at least 4.3 reflectiveunits (RU).
 8. The formulation of claim 4, said total silver content ofsaid at least one silver-containing compound being within a range of0.8% to 3.4%, by weight, the formulation having a standard whitenessvalue of at least 3.5, at least 3.6, at least 3.7, at least 3.8, or atleast 3.9 reflective units (RU).
 9. The formulation of claim 7, at leastone of said aliphatic silver carboxylate and said carrier base selected,such that after standard ultraviolet light (UV) treatment, in which theformulation is subjected to constant exposure to UV for 12 hours at 240nm, said standard whiteness value of the formulation remains within 0.6RU, within 0.5 RU, within 0.4 RU, within 0.3 RU, or within 0.2 RU, of aninitial whiteness value of the formulation prior to said treatment. 10.The formulation of claim 2, at least one of said silver carboxylate andsaid carrier base selected, such that after standard ultraviolet light(UV) treatment, in which the formulation is subjected to constantexposure to UV for 12 hours at 240 nm, a post-UV whiteness value of theformulation remains at least 3.5 reflective units (RU), at least 3.6 RU,at least 3.7 RU, at least 3.8 RU, at least 3.9 RU, at least 4.0 RU, orat least 4.1 RU.
 11. The formulation of claim 9, the formulationcontaining said silver carboxylate in a range of 0.30% to 3.5%, 0.4% to3.5%, 0.5% to 3.5%, 0.6% to 3.5%, 0.7% to 3.5%, 0.30% to 3%, 0.4% to 3%,0.5% to 3%, or 0.6% to 3%, by weight.
 12. The formulation of claim 2,said average valence being at least 1.2, at least 1.3, at least 1.4, atleast 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, orabout 2.0.
 13. The formulation of claim 2, the formulation containingless than 5%, less than 4%, less than 3%, less than 2.5%, less than2.0%, less than 1.5%, less than 1.2%, less than 1%, less than 0.8%, lessthan 0.6%, or less than 0.4% of a whitening agent.
 14. The formulationof claim 2, the formulation containing less than 10%, less than 9%, lessthan 8%, less than 6%, less than 4%, less than 2%, less than 1%, lessthan 0.5%, less than 0.2%, less than 0.1%, or less than 0.05% ofsilver-containing compounds selected from the group consisting ofsilver(II) fluoride (AgF₂), silver(II) picolinate (C₁₂H₈AgN₂O₄),silver(I) oxide and silver(II) oxide.
 15. The formulation of claim 2,said at least one silver carboxylate including an at least divalentsilver carboxylate having a carbon number or average carbon number lessthan 40, less than 38, less than 36, less than 34, less than 33, lessthan 32, less than 31, less than 30, less than 29, less than 28, lessthan 27, less than 25, less than 23, less than 22, or less than
 21. 16.The formulation of claim 2, said at least one silver carboxylateincluding an at least divalent silver carboxylate having a carbon numberor average carbon number greater than 9, greater than 10, or greaterthan
 11. 17. The formulation of claim 2, the formulation containing atleast one carboxylic acid, said at least one carboxylic acid optionallyincluding a corresponding carboxylic acid of said silver carboxylatehaving said nominal valence of 2, a molar ratio of said correspondingcarboxylic acid to said silver carboxylate having said nominal valenceof at least 2 optionally being at least 0.01, at least 0.025, at least0.05, at least 0.1, at least 0.2, at least 0.5, at least 1.0, at least1.5, at least 2, or at least
 3. 18. The formulation of claims 2, ahydrocarbon structure of said carboxylate selected from the groupconsisting of fully saturated, monounsaturated, and polyunsaturatedstructures. 19-21. (canceled)
 22. A method of treating a topicalinfection in need of treatment, comprising applying to said infection atherapeutically effective amount of a formulation comprising: (a) atleast one silver-containing compound, including an anti-microbial agentcontaining an aliphatic silver carboxylate, said silver of saidaliphatic carboxylate having a nominal valence of 2, wherein an averagevalence of silver in said at least one silver-containing compound iswithin a range of 1.1 to 2; and (b) a carrier base; said at least onesilver-containing compound being dispersed within said base, theformulation having a form of a cream, an emulsion, or an ointment, atotal silver content of the formulation being within a range of 0.0005%to 7%, wherein a hydrocarbon structure of said aliphatic silvercarboxylate is selected from the the group consisting of fullysaturated, monounsaturated, and polyunsaturated structures. 23.(canceled)